GSM6022108: BW7 B5 R1; Labrus bergylta; RNA-Seq
Pooled samples kept in RNA later were thoroughly crushed in a mortar kept at -80°C. Fine powder was collected and used to extract total RNA using a tissue homogenizer (Tissue lyser II) and QIAzol reagent® (Invitrogen) including DNase treatment (TURBO DNase, Ambion) according to the manufacturer's protocol. For the first sampling point (BW0), TRIzol reagent® (Invitrogen) was used instead. RNA quality was checked using an Agilent 2100 bio-analyser instrument (Agilent technologies, Waldbronn, Germany). After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.